Revista Brasileira de Farmacognosia 26 (2016) 533–543

www.elsevier.com/locate/bjp

Original Article

Anatomical and histochemical analysis of Dysphania ambrosioides

supported by light and electron microscopy
Rafaela D. Sá a , Asaph S.C.O. Santana a , Flávia C.L. Silva b , Luiz Alberto L. Soares a , Karina P. Randau a,∗
a
Laboratório de Farmacognosia, Departamento de Ciências Farmacêuticas, Universidade Federal de Pernambuco, Recife, PE, Brazil
b
Departamento de Biologia, Universidade Federal Rural de Pernambuco, Recife, PE, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Dysphania ambrosioides (L.) Mosyakin & Clemants (syn: Chenopodium ambrosoides L.), Amaranthaceae,
Received 23 November 2015 popularly known as “mastruz”, is an herb widely used in Brazil as anthelmintic. To contribute to the
Accepted 27 May 2016 knowledge about medicinal plants, a microscopic analysis was accomplished to describe the main
Available online 25 June 2016
anatomical characters of root, stem, petiole and leaf blade of D. ambrosioides and histochemical tests
were performed on the leaf blade. Cross-sections were obtained, by hand, for microscopic analysis of
Keywords: root, stem, petiole and leaf blade; to the leaf blade were still made paradermal sections, scanning electron
Anatomy
microscopy analysis, maceration and histochemical tests. The main characters useful in the identification
Amaranthaceae
Dysphania ambrosioides
of the plant were: anomalous secondary thickening in the root and stem; presence of idioblasts contain-
Histochemistry ing crystal sand in the root, stem, petiole and leaf blade; in these there are also idioblasts with druses;
Mastruz presence of non-glandular and glandular trichomes in the stem, petiole and leaf blade; stomata on the
Microscopic analysis stem, petiole and leaf blade, identified in these as anomocytic and anisocytic; dorsiventral mesophyll and
collateral vascular bundles. Maceration revealed that the vessel elements are helical type. Through the
histochemical tests, it was evidenced the presence of lipophilic substances, essential oils, oleoresins, phe-
nolic compounds, starch, lignin and calcium oxalate crystals. This work provides support to the quality
control of the species.
© 2016 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. This is an open
access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction
The family Chenopodiaceae was comprised about 102 genera
and 1400 species distributed worldwide (Joly, 2002). Recent molec-
ular phylogenetic studies include representatives of the family
Chenopodiaceae in the family Amaranthaceae. Some species of the
genus Chenopodium were transferred to the genus Dysphania, as
Chenopodium ambrosioides L., which is currently known as Dyspha-
nia ambrosioides (L.) Mosyakin & Clemants (Fuentes-Bazan et al.,
2012a,b; Senna, 2016).
D. ambrosioides is popularly known as epazote, Mexican tea,
American wormseed, paico, mastruz and erva-de-Santa-Maria
(Kliks, 1985; Albuquerque et al., 2009). The species is native to
Central and South America, originated, probably, from Mexico. It
has spontaneous growth in tropical and subtropical regions (mainly
America and Africa) and also in temperate zones (from the Mediter-
ranean to Central Europe) (Kismann, 1991). In Brazil, its distribution
∗ Corresponding author.
E-mail: kprandau@ufpe.br (K.P. Randau).
is extensive, occurring in almost all the territory (Sousa et al., 2004;
Lima et al., 2006).
It is an herb that reaches up to 1 m high, being highly branched.
The leaves are alternate, elongated, with jagged edges, acute apex,
hairy and have different sizes, where the smaller are located on
top of the plant and are sessile; the largest stand at the bottom
and have short petiole. They have a strong and characteristic smell.
The inflorescence is the racemosa type, presenting small flowers
green colored. The seeds are numerous, spherical and have black
color (Cruz, 1995; Lorenzi and Matos, 2002; Matos, 2007; Lorenzi,
2008).
The plant is considered by the World Health Organization as
one of the most used among traditional medicines in the world
(Lorenzi and Matos, 2002). The leaves are the part of the plant most
often used in folk medicine as anthelmintic and also as antifungal
(Taylor, 2005; Fenner et al., 2006; Neiva et al., 2011), for digestive
disorders, muscle pains and bone fractures (Santayana et al., 2005;
Garcia et al., 2010). In the Northeast of Brazil, where the species
is widely used, the leaves are mixed in a blender with milk for flu
treatments (Morais et al., 2005). In endemic areas of leishmania-
sis, the population often uses its leaves in the topical treatment of
ulcers caused by the disease (França et al., 1996).

http://dx.doi.org/10.1016/j.bjp.2016.05.010
0102-695X/© 2016 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
534 R. Sá et al. / Revista Brasileira de Farmacognosia 26 (2016) 533–543

Phytochemical studies have identified polyphenols and ter-
penes as the main constituents of D. ambrosioides (Jorge et al., 1986;
Jain et al., 1990; Paré et al., 1993; Kiuchi et al., 2002; Hegazy and
Farrag, 2007; Hallala et al., 2010; Jardim et al., 2010; Neiva et al.,
2011; Okhale et al., 2012; Barros et al., 2013; Sá, 2013). The essential
oil of the leaves is mainly composed of monoterpenes, but the lit-
erature shows that there is wide variation both in its composition
and in its percentage of constituents (Onocha et al., 1999; Gupta
et al., 2002; Cavalli et al., 2004; Jardim et al., 2010; Sá et al., 2014).
Several biological activities have been reported for D. ambro-
sioides (Sá et al., 2015), such as antitumor (Nascimento et al.,
2006; Barros et al., 2013), antipyretic, analgesic, anti-inflammatory,
antinociceptive (Hallala et al., 2010; Trivellato Grassi et al., 2013),
antifungal (Jardim et al., 2010), anthelmintic (Guimaraes et al.,
2001; Neiva et al., 2011) and antiprotozoal (Monzote et al., 2007;
Patrício et al., 2008; Monzote et al., 2014).
Given the recognized popular use and the pharmacological
studies that have demonstrated the therapeutic potential, D.
ambrosioides is one of 71 species of plants that arouse the Brazilian
government’s interest for the production of phytotherapics, being
present in the National Relation Medicinal Plants of Interest to Uni-
fied Health System (MS, 2009).
Considering that the knowledge of the microscopic character-
istics is fundamental for the standardization of plants used as
medicines, this study aims to describe the main anatomical charac-
ters of the root, stem, petiole and leaf blade of D. ambrosioides and
perform histochemical tests on the leaf blade.
Materials and methods
Plant material
Several adult specimens of Dysphania ambrosioides (L.)
Mosyakin & Clemants (syn: Chenopodium ambrosioides L.), Ama-
ranthaceae, cultivated under full sun, were collected in the
garden of the Laboratório de Fitoterapia, in the company
Pernambuco Participações e Investimentos S/A, located in Recife
at the Pernambuco State in Brazil. The voucher specimen was
deposited in the Herbarium UFP-Geraldo Mariz, of the Uni-
versidade Federal de Pernambuco, Brazil, under registration
number 69718.
Anatomical characterization
For their structural characterization, various cross-sections at
the middle region of the root, stem, petiole and leaf blade fixed in
FAA 50% (Johansen, 1940) were obtained by hand, using a com-
mon razor blade. For leaf blade were also performed paradermal
sections on the adaxial and abaxial faces. All sections were clar-
ified in sodium hypochlorite solution (50%) (Kraus and Arduin,
1997). Posteriorly, cross-sections were stained with safranin
and astra blue (Bukatsch, 1972) and paradermal sections were
stained with methylene blue (1%) (Krauter, 1985). Subsequently,
semipermanent histological slides were prepared containing cross-
sections and paradermal sections of botanical material, following
usual plant anatomy procedures (Johansen, 1940; Sass, 1951).
Analyses were performed on images in software (Toup View
Image), obtained by digital camera coupled to a light microscope
(Alltion).
Maceration
The maceration was performed using leaf blade fragments that
were disintegrated with the mixture of 10% nitric acid and 10%
chromic acid (1:1), according to the method of Jeffrey (Johansen,
1940). Analyses were carried out on images in software (Toup View
Image), obtained by digital camera coupled to a light microscope
(Alltion).

cx

pd
pd
cx
vb

A B

vb

id

am

C D

Fig. 1. Dysphania ambrosioides (L.) Mosyakin & Clemants, cross-sections of the root. (A) General aspect, showing peridermis (pd), cortex (cx) and vascular bundle (vb); (B)
Detail of periderm (pd) and cortex (cx); (C) Secondary growth with vascular bundles (vb) irregularly distributed; (D) Detail of amyloplast (am) and idioblast (id) with crystal
sand. Bars: A = 500 ␮m; B, D = 50 ␮m; C = 200 ␮m.
 R. Sá et al. / Revista Brasileira de Farmacognosia 26 (2016) 533–543 535

Histochemical characterization 1940); Dragendorff’s reagent for detecting alkaloids (Farmacopeia

Histochemical tests were made on cross-sections of fresh
leaf blades obtained by the same method used in anatomical
study. The following specific reagents were used to show the
secretion sites and/or accumulation of substances: Sudan III for
lipophilic substances (Sass, 1951); Nadi reagent for essential oils
and oleoresins (David and Carde, 1964); potassium dichromate
(10%) for phenolic compounds (Gabe, 1968); Lugol’s iodine reagent
for starch (Johansen, 1940); phloroglucinol for lignin (Johansen,
Brasileira, 2010); antimony trichloride for triterpenes and steroids
(Mace et al., 1974); vanillin chloridric for tannins (Mace and
Howell, 1974) and hydrochloric acid (10%) to establish the nature
of the crystals (Jensen, 1962).
Controls were performed in parallel with the tests and
semipermanent histological slides were prepared containing the
cross-sections (Johansen, 1940; Sass, 1951). Analyses were per-
formed on images in software (Toup View Image), obtained by
digital camera coupled to a light microscope (Alltion).

tr

co
tr tr
pa vb
id
vb

ep
A B C

tr ep
ep co
id
st pa
id
end

tr vb

D E F

Fig. 2. Dysphania ambrosioides (L.) Mosyakin & Clemants, cross-sections of the stem. (A) General aspect, showing epidermis (ep), trichome (tr), collenchyma (co), parenchyma
(pa), vascular bundles (vb) and idioblast (id) with crystal sand; (B) Detail of non-glandular trichome (tr), multicellular and uniseriate, with enlarged cells at the base and an
elongated apical cell; (C) Detail of non-capitate glandular trichome (tr) with short uniseriate stalk and a small globoid apex and bents toward the epidermis; (D) Detail of
capitate glandular trichome (tr) with a short pedicel and a large unicellular head, stomata (st) and epidermis (ep); (E) Detail of epidermis (ep), trichome (tr), collenchyma
(co), parenchyma (pa), endodermis (end), vascular bundles (vb) and idioblast (id) with crystal sand. (F) Detail of idioblast (id) with crystal sand. Bars: A = 500 ␮m; B, C, D,
F = 50 ␮m; E = 200 ␮m.

ep st tr
co ep
co co

co pa co vb
vb tr
vb
id tr
co

A B C D
ep
co

pa
tr
vb
id

co id
E F vb G H

Fig. 3. Dysphania ambrosioides (L.) Mosyakin & Clemants, cross-sections of the petiole. (A) General aspect, showing epidermis (ep), collenchyma (co), parenchyma (pa),
vascular bundles (vb) and idioblast (id) with crystal sand; (B) Lateral extremity, showing uniseriate epidermis (ep) with stomata (st) and trichome (tr), collenchyma (co),
parenchyma (pa) and vascular bundles (vb); (C) Detail of non-glandular trichome (tr), multicellular and uniseriate, with enlarged cells at the base and an elongated apical
cell; (D) Detail of non-capitate glandular trichome (tr) with short uniseriate stalk and a small globoid apex and bents toward the epidermis; (E) Detail of capitate glandular
trichomes (tr) with a short pedicel and a large unicellular head; (F) Central region, showing epidermis (ep), collenchyma (co), parenchyma (pa), idioblast (id) with crystal
sand and collateral vascular bundles (vb) arranged in a semicircle; (G) Detail of a vascular bundle (vb) located in lateral extremity; (H) Detail of idioblast (id) with crystal
sand. Bars: A = 500 ␮m; B, F = 200 ␮m; C, D, E = 50 ␮m.
536 R. Sá et al. / Revista Brasileira de Farmacognosia 26 (2016) 533–543

Scanning electron microscopy (SEM)
Leaf blades samples were fixed in 2.5% glutaraldehyde (buffered
with 0.1 M sodium cacodylate) and post fixed in 2% osmium
solution (buffered with 0.1 M sodium cacodylate). After
dehydration in ethanol series, the material was submitted to
critical point drying (Bal-Tec CPD 030). Suitable portions were
mounted onto SEM stubs using double-sided adhesive tape and
sputter-coated with gold (Leica EM SCD 500) (Silveira, 1989). Both
adaxial and abaxial surfaces were examined with a QUANTA 200

st
st
ep

crs

ep
A B

tr

tr

C D

tr
tr

E F

tr

dr tr

G H

Fig. 4. Dysphania ambrosioides (L.) Mosyakin & Clemants, frontal view of the leaf blade. (A) View of the adaxial face showing epidermal (ep) cells with straight or slightly
sinuous walls, stomata (st) anomocytic and anisocytic, and idioblast with crystal sand (crs); (B) View of the abaxial face showing epidermal (ep) cells with sinuous walls
and stomata (st) anomocytic and anisocytic; (C) Detail of non-glandular trichome (tr), multicellular and uniseriate, with enlarged cells at the base and an elongated apical
cell on the adaxial face; (D) Detail of non-glandular trichome (tr), multicellular and uniseriate, with enlarged cells at the base and an elongated apical cell on the abaxial
face; (E) Detail of non-capitate glandular trichome (tr) with short uniseriate stalk and a small globoid apex and bents toward the epidermis on the adaxial face; (F) Detail of
non-capitate glandular trichome (tr) with short uniseriate stalk and a small globoid apex and bents toward the epidermis on the abaxial face; (G) Detail of capitate glandular
trichome (tr) with a short pedicel and a large unicellular head on the adaxial face and idioblasts with druses (dr); (H) Detail of capitate glandular trichome (tr) with a short
pedicel and a large unicellular head on the abaxial face. Bars: A–H = 50 ␮m.
 R. Sá et al. / Revista Brasileira de Farmacognosia 26 (2016) 533–543
FEG scanning electron microscope in the Centro de Tecnologias
Estratégicas do Nordeste (CETENE).
Results
Root
In cross-section the root has a circular shape (Fig. 1A) and
presents periderm and a very small cortical region (Fig. 1B), due
to the development of anomalous secondary thickening, character-
ized by a concentric zone of collateral vascular bundles irregularly
distributed, that arise from a succession of arcs of cambium
(Fig. 1C). It is observed in all root several cells containing starch
and idioblasts with crystal sand (Fig. 1D).
Stem
In cross-section, the stem has a polygonal shape, with regions
more prominent (Fig. 2A). Trichomes are located throughout its
extension (Fig. 2A and D), which are non-glandular trichome, mul-
ticellular and uniseriate, with enlarged cells at the base and an
elongated apical cell (Fig. 2B) and also glandular trichomes. These
can be of two types: non-capitate glandular trichome with short
uniseriate stalk and a small globoid apex and bents toward the epi-
dermis (Fig. 2C), and the capitate glandular trichome, with a short
pedicel and a large unicellular head (Fig. 2D).
The epidermis consists of a single layer of cells coated with a
thin cuticle (Fig. 2A, D and E). Stomata are inserted above the level
of the epidermal cells (Fig. 2D). In less prominent regions of the
stem, adjacent to the epidermis, is found a layer of cells that may
be part of the epidermis, making it multilayered, or may constitute
a hypodermis. Already in more prominent regions, the angular col-
lenchyma is located beneath the epidermis, being composed of four
to nine layers of cells (Fig. 2A and E). In the cortical parenchyma are
present idioblasts with crystal sand (Fig. 2A, E and F). The endoder-
mis is seen as a last cortical layer (Fig. 2E). As well as the root,
the stem also exhibits anomalous secondary thickening. It is dis-
tinguished two different zones of vascular bundles: a zone closer
to the endodermis, in which the collateral bundles are distributed
forming a continuous ring; and other zone closest to the medullary
region, in which the collateral bundles are distributed discontin-
uously, separated from each other by parenchyma (Fig. 2A and
E).
Petiole
The petiole, in cross-section, has concave–convex shape, with
two lateral extremities (Fig. 3A). The epidermis is composed of
a single layer of rounded cells and covered with a smooth and
tr tr
st
tr
ep
ep
A B
Fig. 5. Dysphania ambrosioides (L.) Mosyakin & Clemants, SEM of the leaf blade. (A) View of the abaxial face showing epidermal (ep) cells with sinuous walls, stomata (st)
and non-glandular and glandular trichomes (tr); (B) View of the abaxial face showing epidermal (ep) cells with sinuous walls, stomata (st) and glandular trichomes (tr); (C)
View of the adaxial face showing epidermal (ep) cells with slightly sinuous walls, stomata (st) and glandular trichome (tr). Bars: A, B = 100 ␮m; C = 40 ␮m.
537
thin cuticle (Fig. 3A and B). Stomata are inserted in the same
level of epidermal cells (Fig. 3B). As epidermal attachments, are
present non-glandular trichome, multicellular and uniseriate, with
enlarged cells at the base and an elongated apical cell (Fig. 3C),
besides non-capitate glandular trichome with short uniseriate
stalk and a small globoid apex and bents toward the epidermis
(Fig. 3D) and capitate glandular trichome, with a short pedicel and
a large unicellular head (Fig. 3E). The collenchyma of the angu-
lar type is observed below the epidermis in the central region of
the petiole (Fig. 3A and F), being formed by two or three layers
of cells. This tissue is also present in the lateral extremities and
adjacent to phloem (Fig. 3A, B and F). The vascular bundles are
collateral and are arranged in a semicircle in the central region
of the petiole (Fig. 3A and F). Smaller vascular bundles are dis-
played in the lateral extremities (Fig. 3B and G). In the parenchyma
are observed idioblasts containing crystal sand (Fig. 3A, F
and H).
Leaf blade
The epidermis, in frontal view, consists of cells with straight or
slightly sinuous walls on the adaxial face (Fig. 4A) and of cells with
walls more sinuous on the abaxial face (Fig. 4B). The stomata, that
are anomocytic and anisocytic, are present on both sides (Fig. 4A
and B). Non-glandular trichomes (Fig. 4C and D) and glandular tri-
chomes (Fig. 4E–H) occur both on the adaxial face (Fig. 4E and G)
as on the abaxial face (Fig. 4F and H) and are of the same type pre-
viously described in the stem and petiole. However, the glandular
trichomes occur in greater number on the adaxial face, among the
two types of glandular trichomes found, predominate the capitate
glandular trichome. In this cut is still possible to view idioblasts
with crystal sand (Fig. 4A) and druses (Fig. 4G).
In the leaf blade analysis in SEM it was possible observed with
more detail the largest sinuosity of the epidermal cells walls on
the abaxial face (Fig. 5A and B) and that on this face the stomata
are situated on the same level or slightly above of epidermal cells
(Fig. 5B), while on the adaxial face they are located on the same level
of epidermal cells (Fig. 5C). It was also observed the non-glandular
(Fig. 5A) and glandular trichomes (Fig. 5A–C).
In cross-section of the leaf blade were observed all the trichomes
(Fig. 6A–F) viewed in the paradermal cut and in SEM, also ratifying
the fact that the glandular trichomes are predominant in the abaxial
face. The epidermis is uniseriate, coated with a thin cuticle layer and
is made of rounded cells or slightly elongated (Fig. 6G). The meso-
phyll has organization dorsiventral. It consists of one or two layers
of palisade parenchyma and two to four layers of dense spongy
parenchyma (Fig. 6G). Secretory cavities (Fig. 6G) and idioblasts
with crystal sand and druses (Fig. 6G and H) are found in the meso-
phyll.
st ep
tr
tr
st
C
538 R. Sá et al. / Revista Brasileira de Farmacognosia 26 (2016) 533–543

tr

tr
A B

tr

C D tr

tr

tr
E F

pp ep
dr

sp crs

scv
vb

G ep H

Fig. 6. Dysphania ambrosioides (L.) Mosyakin & Clemants, cross-sections of the leaf blade. (A) Detail of non-glandular trichome (tr), multicellular and uniseriate, with enlarged
cells at the base and an elongated apical cell on the adaxial face; (B) Detail of non-glandular trichome (tr), multicellular and uniseriate, with enlarged cells at the base and
an elongated apical cell on the abaxial face; (C) Detail of non-capitate glandular trichome (tr) with short uniseriate stalk and a small globoid apex and bents toward the
epidermis on the adaxial face; (D) Detail of non-capitate glandular trichome (tr) with short uniseriate stalk and a small globoid apex and bents toward the epidermis on the
abaxial face; (E) Detail of capitate glandular trichome (tr) with a short pedicel and a large unicellular head on the adaxial face; (F) Detail of capitate glandular trichome (tr)
with a short pedicel and a large unicellular head on the abaxial face; (G) Detail of the epidermis (ep), palisade parenchyma (pp), spongy parenchyma (sp), secretory cavity
(scv), vascular bundle (vb) and idioblast with druse (dr); (H) Detail of idioblast with crystal sand (crs). Bars: A–H = 50 ␮m.

The midrib has biconvex cross-section (Fig. 7A). It presents
similar epidermis to which is situated in the mesophyll (Fig. 7B).
Furthermore, all three types of trichomes visualized in the
mesophyll are also present in the midrib (Fig. 7C–E). Angular col-
lenchyma appears in subepidermal position, formed by two to three
layers of cells (Fig. 7A and B), and also adjacent to phloem (Fig. 7A
and F). In this tissue and in the parenchyma are observed idioblasts
with crystal sand and druses (Fig. 7B and F). The vascular system is
composed of collateral bundles arranged in a circle in the center of
midrib (Fig. 7F).
 R. Sá et al. / Revista Brasileira de Farmacognosia 26 (2016) 533–543 539

co
ep

tr pa co
co crs
vb dr
tr

A co B C

crs

dr

tr pa

vb
tr co
D E F

Fig. 7. Dysphania ambrosioides (L.) Mosyakin & Clemants, cross-sections of midri(B) (A) General aspect, showing collenchyma (co), parenchyma (pa), vascular bundles (vb) and
trichome (tr); (B) Detail of the epidermis (ep), collenchyma (co) and idioblasts with crystal sand (crs) and druse (dr); (C) Detail of non-glandular trichome (tr), multicellular
and uniseriate, with enlarged cells at the base and an elongated apical cell; (D) Detail of non-capitate glandular trichome (tr) with short uniseriate stalk and a small globoid
apex and bents toward the epidermis; (E) Detail of capitate glandular trichome (tr) with a short pedicel and a large unicellular head; (F) Detail of the vascular bundles (vb)
located in the parenchyma (pa), collenchyma (co) adjacent to phloem and idioblasts with crystal sand (crs) and druse (dr). Bars: A = 500 ␮m; B, C, D, E = 50 ␮m; F = 200 ␮m.

The leaf of D. ambrosioides, after maceration, presents epidermal
cells, stomata (Fig. 8A), vessel elements of the helical type (Fig. 8B)
and idioblasts with druses (Fig. 8C). The three types of trichomes
viewed in transverse and paradermal sections and in SEM are also
found in macerated leaf (Fig. 8D–F).
Fig. 9A–D shows cross-sections of fresh leaf blades without addi-
tion of reagent.
After using Sudan III, lipophilic substances were found in the
cuticle covering the adaxial and abaxial faces (Fig. 10A), and are
also present within capitate glandular trichomes with unicel-
lular head (Fig. 10B). In these trichomes, lipophilic substances
may be those which constitute the essential oil and oleoresins of
D. ambrosioides, considering that, with the Nadi reagent, these com-
pounds exhibit blue and pink colorations, respectively (Fig. 10C and
D). Oleoresins were also found in the parenchyma cells of the midrib
(Fig. 10E) and in the upper epidermis cells next to the mesophyll
(Fig. 10F).
Potassium dichromate (10%) revealed the presence of pheno-
lic compounds in the adaxial epidermal cells (Fig. 11A and B), as
well as inside the capitate glandular trichome with unicellular head
(Fig. 11C). Starch is present in the epidermal cells and in the meso-
phyll (Fig. 11D), as also in the parenchyma of the midrib (Fig. 11E).
The phloroglucinol evidenced lignification of xylematic vessel in
midrib (Fig. 11F). Fig. 11G and H show, respectively, the presence

dr
st dr
ep

A B C

tr

tr
tr

D E F

Fig. 8. Dysphania ambrosioides (L.) Mosyakin & Clemants, maceration of the leaf. (A) Epidermis (ep) and stomata (st); (B) Vessel element of the helical type; (C) Idioblasts with
druses (dr); (D) Non-glandular trichome (tr), multicellular and uniseriate, with enlarged cells at the base and an elongated apical cell; (E) Non-capitate glandular trichome
(tr) with short uniseriate stalk and a small globoid apex and bents toward the epidermis; (F) Capitate glandular trichome (tr) with a short pedicel and a large unicellular
head. Bars: A = 200 ␮m; B–F = 50 ␮m.
540 R. Sá et al. / Revista Brasileira de Farmacognosia 26 (2016) 533–543

ep
co

vb vb

pa

A B

ct
ep
tr
pp

sp

C D

Fig. 9. Dysphania ambrosioides (L.) Mosyakin & Clemants, cross-sections of the leaf blade without adding reagents. (A) Midrib showing epidermis (ep), collenchyma (co),
parenchyma (pa) and vascular bundles (vb); (B) Detail of vascular bundle (vb); (C) Details of cuticle (ct), epidermis (ep), palisade parenchyma (pp) and spongy parenchyma
(sp); (D) Detail of capitate glandular trichome (tr) with a short pedicel and a large unicellular head. Bars: A = 200 ␮m; B–D = 50 ␮m.

ct
tr

tr
ct
A B C

pa
tr

ep

D E F

Fig. 10. Dysphania ambrosioides (L.) Mosyakin & Clemants, cross-sections of the leaf blade after reaction with Sudan III and Nadi reagent. (A) Lipophilic substances in the
cuticle (ct) lining the epidermis; (B) Lipophilic substances inside capitate glandular trichome (tr); (C) Essential oil inside capitate glandular trichome (tr); (D) Oleoresin inside
capitate glandular trichome (tr); (E) Oleoresin in parenchyma (pa); (F) Oleoresin in epidermal (ep) cells. Bars: A–F = 50 ␮m.

of crystals in leaf of D. ambrosioides and their dissolution with the
test of hydrochloric acid (10%), confirming that they are of calcium
oxalate. Tests with Dragendorff’s reagent, antimony trichloride and
vanillin chloridric were negative.
Discussion
The transition of some species of the family Chenopodiaceae to
the family Amaranthaceae is recent. Although the modification, the
literature still treats D. ambrosioides as C. ambrosioides. For this rea-
son, the discussion of this work took into consideration the aspects
of the family Chenopodiaceae.
According to Metcalfe and Chalk (1972), the family Chenopodi-
aceae has many anatomical points in common with other families,
such as the Amaranthaceae. One of these points is the cambium
variation, or anomalous secondary thickening (Wilson, 1924; Joshi,
1937; Balfour, 1965). The formation of successive cambia is known
in 34 species of dicotyledons, being that in lianas this phenomenon
 R. Sá et al. / Revista Brasileira de Farmacognosia 26 (2016) 533–543 541

ep ep
pp
ep
sp

tr

A B C D

crs id
pa
vb

E F G H
Fig. 11. Dysphania ambrosioides (L.) Mosyakin & Clemants, cross-sections of the leaf blade after reaction with potassium dichromate (10%), Lugol’s iodine reagent, phloroglu-
cinol and hydrochloric acid (10%). (A) View of the leaf showing phenolic compounds in epidermal (ep) cells; (B) Detail of epidermal (ep) cells containing phenolic compounds;
(C) Detail of phenolic compounds inside capitate glandular trichome (tr); (D) Starch in epidermal (ep) cells, palisade parenchyma (pp) and spongy parenchyma (sp); (E) Detail
of starch in parenchyma (pa); (F) View of vascular bundles (vb) with lignified walls; (G) Detail of idioblast with crystal sand (crs) before reaction with hydrochloric acid (10%);
(H) Detail of idioblast (id) after reaction with hydrochloric acid (10%). Bars: A = 200 ␮m; B–H = 50 ␮m.

occurs more frequently (Metcalfe and Chalk, 1972; Carlquist, 2007).
In Chenopodiaceae, the secondary growth starts from a cambium in
a normal position. Subsequently, the other cambia emerge furthest
from the first, producing xylem to inside and phloem to outside
(Esau, 1997). The successive cambia and the vascular bundles are
embedded in parenchymal tissue (Metcalfe and Chalk, 1972).
With respect yet to the secondary growth of the plant, it is
observed the periderm formation in roots, but not in stems. The
absence of periderm in stem was already observed in 12 species
of Chenopodium, including D. ambrosioides (Bonzani et al., 2003), as
well as in other species of Chenopodiaceae, such as Atriplex halimus,
Atriplex cristata, Atriplex oestophora (Fahn and Zimmermann, 1982;
Jáuregui et al., 2014); Allenrolfea patagonica, Heterostachys olivas-
cens, Heterostachys ritteriana (Cuadra and Hermann, 2014); and in
Salsola kali subsp. Ruthenica (Bercu and Bavaru, 2004).
The polygonal shape of the stem with the presence of regions
more prominent formed by collenchymatic tissue is common in
Chenopodiaceae (Fahn and Zimmermann, 1982; Bonzani et al.,
2003; Bercu and Bavaru, 2004; Jáuregui et al., 2014). Bonzani et al.
(2003) observed that in Chenopodium burkartii and Chenopodium
retusum the collenchyma becomes lignified when these species are
in secondary growth. Already in other species of Chenopodium stud-
ied, including D. ambrosioides, the collenchyma remains composed
of cellulosic walls in secondary growth.
Stomata were found in the stem, petiole and leaf blade of the
plant. Amphistomatic leaves are characteristics of the Chenopo-
diaceae family (Metcalfe and Chalk, 1972; Moris et al., 1996;
Bonzani et al., 2003; Cuadra and Hermann, 2014; Jáuregui et al.,
2014). Anomocytic stomata are the most frequent, but are also
found anisocytic stomata in Chenopodium chilense, tetracytic in
Chenopodium multifidum (Bonzani et al., 2003) and paracytic in Sal-
sola kali subsp. ruthenica (Bercu and Bavaru, 2004). Previous studies
with D. ambrosioides only affirmed the presence of anomocytic
stomata, different from the results found in this study, which are
described stomata anomocytic and anisocytic (Jorge et al., 1986;
Costa and Tavares, 2006).
The presence of different types of trichomes in Chenopodiaceae
was reported by some authors (Holm, 1923; Metcalfe and Chalk,
1972; Silva Filho et al., 1992; Bonzani et al., 2003). According to
Metcalfe and Chalk (1972), in the family are present trichomes
uniseriate, branched, stellate and capitate glandular trichome.
In the genus Chenopodium are most common the non-glandular
trichome uniseriate and the capitate glandular trichome. Both
were observed in the stem, petiole and leaf blade of D. ambrosioides,
but it was also found the glandular trichome uniseriate with body
bent toward epidermis and with secretory distal cell rounded.
This latter type of trichome is not described by Metcalfe and Chalk
(1972), however, in a detailed study of the trichomes of the stem
and leaves of Chenopodium species, Bonzani et al. (2003) cited all
kinds of trichomes found in this work.
There is still controversy regarding the presence of glandular
trichomes on the faces of the leaf blade of D. ambrosioides. As are
seen in this study, the presence on both faces was also identified
by Holm (1923), Jorge et al. (1986) and Bonzani et al. (2003) in
D. ambrosioides. Jorge et al. (1986) affirmed that the glandular tri-
chomes predominate in the adaxial face, in agreement with the
results described in this paper and differing from Costa and Tavares
(2006), who reported these trichomes restricted to abaxial face.
Studies suggest that the density and the change in the propor-
tion of non-glandular and glandular trichomes can be influenced
by environmental conditions, including herbivory and water avail-
ability (Rautio et al., 2002; Gonzales et al., 2008). Since the species
is found in tropical, subtropical and temperate zones (Kismann,
1991), it can be expected that these variations occur in D. ambro-
sioides.
Most species of Chenopodiaceae have dorsiventral mesophyll
(Metcalfe and Chalk, 1972). There are some exceptions, such as
C. retusum, Chenopodium oblanceolatum (Bonzani et al., 2003) and
Salsola sp. (Metcalfe and Chalk, 1972) that present isobilateral
mesophyll.
Idioblasts containing crystal sand were often found in all plant
parts examined. Only in the leaf blade were observed druses. The
confirmation, by testing with hydrochloric acid that the crystals
are of calcium oxalate corroborates the result of Costa and Tavares
(2006). According Metcalfe and Chalk (1972), the occurrence of
these crystals gives important diagnostic value for the species, since
they are restricted between dicotyledonous.
The maceration of plant tissue is used to reveal some pecu-
liarities of the nature of the cells that compose them. It is a
technique recommended by the Brazilian Pharmacopeia for micro-
scopic analysis of the plant material (Farmacopeia Brasileira, 2010)
and a requirement for the registration of phytotherapic and tradi-
tional phytotherapic products (Anvisa, 2014). It is also important
when the plant raw materials are marketed crushed or powdered,
542 R. Sá et al. / Revista Brasileira de Farmacognosia 26 (2016) 533–543
not being possible performing sections to the anatomical study
(Farmacopeia Brasileira, 2010).
They were detected in the leaf blades lipophilic and hydrophilic
substances, evidencing lipids, essential oils, oleoresins and phe-
nolic compounds. These data corroborate the findings in the
literature for the species, known for producing various compounds
of metabolism used in the defense and adaptation of plants to the
environment and also in medicine (Sá et al., 2015). Most plants
store starch as a reserve. This carbohydrate, as well as being related
as an energy source is also an adaptive strategy of the species to
adverse environmental conditions (Oliveira and Marquis, 2002).
The lignin present in the wall of the xylem vessels is responsible
for sustaining the plant and also provides defense for the plant,
because it is considered as a substance resistant to pathogens,
hidering their colonization (Silva et al., 2005).
Conclusion
Through the different microscopy techniques it was possible to
establish anatomical features that are useful in the identification of
D. ambrosioides, which were: anomalous secondary thickening in
the root and stem; presence of idioblasts containing crystal sand
in the root, stem, petiole and leaf blade; in these there are also
idioblasts with druses; presence of non-glandular and glandular
trichomes in the stem, petiole and leaf blade; stomata on the stem,
petiole and leaf blade, identified in that as anomocytic and aniso-
cytic; dorsiventral mesophyll and collateral vascular bundles. The
maceration showed to be a useful tool when it cannot make cuts
to the anatomical study. In addition to contributing significantly
to the knowledge of the anatomy, it was also possible to see the
histolocalization of some groups of metabolites present in the leaf
blade of the species. Thus, the work provides quality parameters for
the species studied, since it does not have appropriate monograph
in current official codes.
Authors’ contributions
RS contributed in collecting plant sample and identification,
confection of herbarium, running the laboratory work, analysis of
the data and drafted the paper. AS contributed in the preparation
of semi-permanent slides. FS and LS contributed to critical reading
of the manuscript. KR designed the study, supervised the labora-
tory work and contributed to critical reading of the manuscript.
All the authors have read the final manuscript and approved the
submission.
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgments
The authors are grateful to CNPq (132722/2011-9) for financial
support in the form of grants and fellowship awards. They also
thank to PERPART for supplying the plant material.
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